Liposomes are composed of at least one lipid bilayer membrane enclosing an aqueous internal compartment. Liposomes may be characterized by membrane type and by size. Small unilamellar vesicles (SUVs) have a single membrane and typically range between 0.02 and 0.25 μm in diameter; large unilamellar vesicles (LUVs) are typically larger than 0.25 μm. Oligolamellar large vesicles and multilamellar large vesicles have multiple, usually concentric, membrane layers and are typically larger than 0.25 μm. Liposomes with several nonconcentric membranes, i.e., several small vesicles contained within a larger vesicle, are termed multivesicular vesicles.
Liposomes may be formulated to carry therapeutic agents, drugs or other active agents either contained within the aqueous interior space (water soluble active agents) or partitioned into the lipid bilayer (water-insoluble active agents). Liposomes may also be conjugated to an antibody or targeting molecule that permits the delivery of active agent to a specific target site. Encapsulation of a drug in a liposome (1) reduces toxicity of the drug, (2) avoids the body's defenses that normally recognize foreign particles and target them for removal by the reticuloendothelial system (RES) of the liver and spleen, and (3) allows targeting of the drug carrier to the therapeutic site of action, and once there, to release the drug rapidly so that it can act on the target tissue. Further, clearance of the liposome from blood by the cells of the reticuloendothelial system (RES) can be inhibited by incorporating polyethyleneglycol lipids into the liposome membrane; these lipids inhibit the protein adsorption that labels the liposome for RES uptake.
Liposomes can be designed to be not leaky but will become so if a pore occurs in the liposome membrane, or if the membrane becomes fluid (e.g. undergoes a phase transition from a solid or gel phase to a liquid phase), or if the membrane degrades or dissolves. Such a breakdown in permeability can be induced by the application of electric fields (electroporation), or exposure of the liposome to enzymes or surfactants. Another method involves raising the temperature of the membrane to temperatures in the vicinity of its gel to liquid phase transition temperature, where it appears that porous defects at phase boundary regions in the partially liquid and partially solid membrane allow for increased transport of water, ions and small molecules across the membrane. The clinical elevation of temperature in the body is called hyperthermia. This procedure has been used to raise the temperature at a target site in a subject and if temperature-sensitive liposomes can be delivered to the target site then this increase in temperature can trigger the release of liposome contents, giving rise to the selective delivery and release of therapeutic agents at the target site, as initially described by Yatvin et al., Science 204:188 (1979). This technique is limited, however, to conditions where the phase transition temperature of the liposome is higher (greater than 37° C.) than the normal tissue temperature.
Hyperthermia causes multiple biologic changes. For a review refer to Issels R D. Hyperthermia adds to Chemotherapy, European J of Cancer (2008) 44:2546-2554. Temperatures in the mild hyperthermia range (39-44° C.) mediate localized physiological changes such as increases in blood flow, vasculature permeability and tissue oxygenation. The vasculature supporting solid tumors is chaotic in structure and the endothelial cells lining the micro-vasculature do not seal together normally resulting in a porous quality. Hyperthermia causes an increase in the pore size in the abnormal tumor microvasculature and therefore enhances the extravasation of nanoscale molecules, such as liposomes of about 100 nm diameter, into the tumor interstitium (Bates D A, Mackillop W J. Hyperthermia, adriamycin transport, and cytotoxicity in drug-sensitive and -resistant Chinese hamster ovary cells, Cancer Res (1986) 46:5477-5481; Nagaoka S, Kawasaki S, Sasaki K, Nakanishi T. Intracellular uptake, retention and cytotoxic effect of adriamycin combined with hyperthermia in vitro. Jpn J Cancer Res (1986) 77:205-211). For these reasons mild hyperthermia is selectively lethal to tumor cells, with the antitumor effect increasing as the temperature increases.
Heat sensitive liposomes carry a high concentration of chemotherapeutic agent to solid tumors and the supporting vasculature and release drug locally when heated. Hyperthermia selectively increases liposomal uptake, liposomal permeability, stimulates localized drug release, increases the influx of drug into tumor cells, and increases drug binding to tumor cell DNA (the latter being essential to the mechanism of action of a number of chemotherapeutic agents).
In order to begin to use hyperthermia for the treatment of deep-seated tumors (e.g., prostate, ovarian, colorectal and breast tumors), it is accordingly desirable to devise liposome formulations capable of delivering therapeutic amounts of active agents in response to mild hyperthermic conditions, i.e., for clinically attainable temperatures in the range 39-45° C.
U.S. Pat. No. 6,726,925 describes liposomes that are sensitive to alterations in the temperature of the surrounding environment. The liposomes are loaded with, inter alia, doxorubicin, an approved and frequently used oncology drug for the treatment of a wide range of cancers. The doxorubicin containing liposomal formulation is administered intravenously and in combination with hyperthermia can provide local tumor control and improve quality of life. Localized mild hyperthermia (39.5-45 degrees Celsius) releases the entrapped doxorubicin from the liposome. This delivery technology enables high concentrations of doxorubicin to be deposited preferentially in a targeted tumor. U.S. Pat. No. 7,901,709 describes a method for heat-activated liposomal encapsulation of doxorubicin.
Published International Application No. WO 2007/024826 describes a method of storing a liposome or nanoparticle formulation including freezing such a formulation. The formulation describes a method of storing liposomes having enhanced stability and storage characteristics.
Key design principles that are required for a hyperthermically activated liposomal-drug formulation to be effective are: 1) near complete encapsulation of active agent to allow the drug to be associated with the liposome in the systemic circulation, 2) a membrane that is engineered to retain drug at normal body temperatures (37° C.) and release drug at mild hyperthermia temperatures (i.e. 41-43° C.), 3) a membrane composition and particle size that allows the liposome to remain in the systemic circulation long enough to allow the application of a heating modality to trigger the release of the drug to its target, and 4) liposome size that permits its extravasation from the blood stream across leaky tumor micro-vasculature permitting targeting of chemotherapeutic drugs to a tumor site.
An additional important design issue discovered by the inventors with liposomal formulations of doxorubicin (e.g. disclosed in U.S. Pat. No. 7,901,709) is the stabilizing effect of doxorubicin complex (co-crystal or salt) formation on the stability of the finished drug product. Successful control of degradation rates will result in significant impact on the storage temperature and long term stability of the drug product. Two degradation products of interest are 8-desacetyl-8-carboxy daunorubicin and impurity A.
The present invention solves a persistent problem with drug degradation in doxorubicin liposomal formulation that results in a citrate complex (co-crystal or salt). It has been found that the citrate complex plays a significant role in doxorubicin instability and formation of degradates. More specifically, the formation of 8-desacetyl-8-carboxy daunorubicin and impurity A can be significantly reduced by changing the formation of a citrate complex to a sulfate complex (co-crystal or salt). In addition, the present invention maintains the key design principles listed above for an efficacious hyperthermically activated liposomal formulation containing an active agent.